A. Sequence-Specific DNA-Binding Molecules And Diseases
Because of their involvement in disease development and progression, determining the presence and activity of sequence-specific DNA-binding molecules is of great importance in the diagnosis, and treatment of disease. For example, sequence-specific DNA-binding molecules that are involved in the development and progression of cancer are potential targets for anti-cancer drugs. In particular, the estrogen-receptor is a sequence-specific DNA-binding protein that is required for the growth of certain breast tumors. Detection of the sequence-specific DNA binding activity of the estrogen receptor in breast tumor cells allows rational selection of therapeutic drugs (e.g., tamoxifen) that bind to the estrogen-receptor and inhibit its ability to bind to DNA. Additionally, the availability of assays for the sequence-specific DNA binding activity of the estrogen-receptor is valuable in the discovery, screening and development of anti-estrogen drugs.
Similarly, assays for other sequences-specific DNA-binding molecules (e.g., hormone receptors) are also valuable for screening the sequence-specific DNA binding activity of anti-testosterone drugs for the treatment of prostate cancer. Assays for the sequence-specific DNA binding hormone receptor for cortisone are also useful since cortisone is an anti-inflammatory agent that potentiates anti-cancer chemotherapy by reducing the levels of NF.kappa.B and of other sequence-specific DNA-binding molecules that, among other actions, protect cancer cells from programmed cell death (apoptosis). Assays of sequence-specific DNA binding activity of molecules (e.g., RNA polymerases, TATA box binding protein) that are targeted by antiviral and antibacterial drugs are also useful in developing and screening candidate drugs.
B. Current Assays
Currently available methods for measuring, the presence of sequence-specific DNA-binding molecules include immunological assays, DNA-binding assays, and expression assays. In immunological assays (e.g. fluorescence microscopy, ELISA, Western blotting), an antibody or antiserum is produced using the purified sequence-specific DNA-binding molecule. The antibodies are then used to detect the presence of the sequence-specific DNA-binding molecule in test samples. While these methods are specific and sensitive, they require several hours or days to complete. Importantly, immunological methods suffer from the drawback that they determine only the presence, not the sequence-specific DNA binding activity, of a sequence-specific DNA-binding molecule.
DNA-binding assays include gel retardation assays and DNA footprinting. In gel retardation assays, a double stranded synthetic oligonucleotide is constructed, having the specific nucleotide sequence to that the sequence-specific DNA-binding protein binds. The oligonucleotide is labeled, usually with radioactive phosphate, and incubated with the preparation containing the DNA-binding protein. The oligonucleotide is then electrophoresed on a suitable gel. The binding of a sequence-specific DNA-binding protein is detected as a retardation in the migration of the radioactive oligonucleotide. In DNA "foot printing" assays, a radioactively-labeled DNA molecule containing the DNA sequence to that the sequence-specific DNA-binding molecule binds is incubated with the sequence-specific DNA-binding molecule and then digested with DNAaseI, an enzyme that cuts DNA molecules regardless of their sequence. This forms DNA fragments of all possible lengths that can be separated by sequencing gel electrophoresis. A "ladder" of these different length fragments is formed on the gel. Binding of the sequence-specific DNA-binding molecule to its cognate nucleotide recognition sequence protects the DNA in that region from digestion with DNAaseI. This protection is observed as a region of reduced intensity of radioactivity on a sequencing gel, i.e., the "ladder" is missing, contiguous "rungs" where the protein was bound.
While DNA-binding assays are specific and sensitive, they nonetheless are laborious, requiring 1 to 7 days to perform, and are technically difficult. In addition, the gel retardation assay is suitable only for use with proteins that bind the DNA with very high affinity, since this assay requires that the protein remains bound to the DNA under the condition of extreme dilution used during gel electrophoresis. More importantly, both gel retardation assays and DNA footprinting assays are not quantitative.
Expression assays are directed to measuring the activity of sequence-specific DNA-binding molecules that function as gene regulators. This activity is assessed by measuring the ability of the gene regulator to induce or suppress the production of a specific mRNA or protein. This is done by inserting the nucleotide sequence upstream of a "reporter" gene and introducing the resulting vector into cells or into a suitable cell free in vitro system. The addition of the gene regulator modifies expression of the reporter gene. Expression assays are technically complex, time consuming (require several days or weeks to perform), and are not quantitative.
Other methods are available that measure the presence and/or activity of particular sequence-specific DNA-binding molecules. For example, the activity of the estrogen receptor in binding estrogen may be measured using radioactive estrogen. Similarly, RNA polymerase activity in initiating DNA transcription may be measured by quantitating the amount of RNA produced from a DNA template. Although these methods may be sensitive, specific, and quantitative for the particular molecule whose activity they are designed to measure, they are nevertheless not universally applicable to any sequence-specific DNA-binding molecule.
C. RNA Polymerases
RNA polymerases constitute a family of enzymes that transcribe DNA sequences into complementary RNA molecules. Because of the universality of the role of RNA polymerases in transcription in all cell types, the availability of assays to measure transcription activity of RNA polymerases is a valuable tool both in basic and applied research, and in the screening of inhibitors of RNA polymerases as antibacterial, antiviral, and anti-cancer drugs.
Currently available assays for RNA polymerase transcription activity measure production of RNA molecules. Transcription of specific genes is assayed in vitro by measuring, the incorporation of radioactive uridine into transcribed RNA molecules that are produced from a defined DNA template in the presence of cell extracts containing RNA polymerase and all the required transcription factors. Transcription of specific genes can also be performed in cell cultures by detecting the production of specific mRNAs (e.g., by Northern blots) or by detecting the production of specific proteins (e.g., by immunoassays). Currently available assays for RNA polymerase transcription activity suffer from the disadvantages that they are laborious, complex, and require the use of radioactive isotopes.
Thus what is needed are compositions and methods for detecting and determining the DNA-binding activity of sequence-specific DNA-binding molecules. Also needed, are compositions and methods for detecting and determining the activity of RNA polymerases in initiating transcription.